Heme proteins encompass a wide range of functions that include electron transfer, transport and storage of oxygen, production and sensing of nitric oxide, decomposition of reactive oxygen species, catalytic oxidation of substrates, signal transduction and control of gene expression (Gray et al., “Electron Transfer in Engineered Heme Enzymes,” Faseb. J. 11:A781-A781 (1997); Sono et al., “Heme-Containing Oxygenases,” Chem. Rev. 96:2841-2888 (1996); Alderton et al., “Nitric Oxide Synthases Structure, Function and Inhibition,” Biochem. J. 357:593-615 (2001); and Perutz et al., “A Haemoglobin that Acts as an Oxygen Sensor: Signalling Mechanism and Structural Basis of its Homology with PAS Domains,” Chem. Biol. 6:R291-R297 (1999)). Under incorporation of heme into recombinant proteins can adversely influence their characterization. Evaluation of enzymatic activity is systematically underestimated if a significant proportion of the recombinant sample does not contain heme. Furthermore, proteins devoid of heme may bind another non-native cofactor with a contaminating activity of its own. A pure protein sample is usually essential for structural characterization by techniques such as X-ray crystallography, as this method often requires a single, well-folded species for crystallization. Other methods of heme protein characterization are less sensitive to the level of heme incorporation, particularly if those methods directly detect the metallocofactor (e.g., UV/Vis, EPR, and Mossbauer spectroscopy) or rely on enzymatic activity. This is both good and bad in that under incorporation may not greatly affect the analysis, but heterogeneity in the sample may go undetected.
Incomplete heme incorporation into recombinant proteins has been a frequently encountered problem (Varadarajan et al., “Cloning, Expression in Escherichia-Coli, and Reconstitution of Human Myoglobin,” Pro. Nat. Acad. Sci. U.S.A. 82:5681-5684 (1985); Ishikawa et al., “Expression of Rat Heme Oxygenase in Escherichia-Coli as a Catalytically Active, Full-Length Form that Binds to Bacterial-Membranes,” Eur. J. Biochem. 202:161-165 (1991); Smith et al., “Expression of a Synthetic Gene for Horseradish Peroxidase-C in Escherichia-Coli and Folding and Activation of the Recombinant Enzyme with Ca-2+ and Heme,” J. Biol. Chem. 265:13335-13343 (1990); Kery et al., “Delta-Aminolevulinate Increases Heme Saturation and Yield of Human Cystathionine Beta-Synthase Expressed in Escherichia-Coli,” Archives Biochem. Biophys. 31:624-29 (1995); Varnado et al., “Properties of a Novel Periplasmic Catalase-Peroxidase from Escherichia Coli O157: H7,” Archives Biochem. Biophys. 42:1166-174 (2004); and Graves et al., “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta-Proteins And Proteomics 1784:1471-1479 (2008)) and thus, techniques have been developed to improve heme loading (Kery et al., “Delta-Aminolevulinate Increases Heme Saturation and Yield of Human Cystathionine Beta-Synthase Expressed in Escherichia-Coli,” Archives Biochem. Biophys. 31:624′29 (1995); Varnado et al., “Properties of a Novel Periplasmic Catalase-Peroxidase from Escherichia Coli O157: H7,” Archives Biochem. Biophys. 42:1166-174 (2004); Graves et al., “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta-Proteins And Proteomics 1784:1471-1479 (2008); Weickert et al., “Optimization of Heterologous Protein Production in Escherichia Coli,” Curr. Opinion In Biotechnol. 7:494-499 (1996); Shen et al., “Production of Unmodified Human Adult Hemoglobin in Escherichia-Coli,” Pro. Nat, Acad. Sci. U.S.A. 90:8108-8112 (1993); Varnado et al., “System for the Expression of Recombinant Hemoproteins in Escherichia Coli,” Prot. Exp. Pur. 35:76-83 (2004); and Vaniado et al., “Expression of Recombinant Hemoproteins in E. Coli Using a Heme Protein Expression System,” Biophys. J. 384A-384A (2007)). During induction of recombinant protein expression from highly active vectors, such as those that employ the T7-polymerase, a population of protein will fold without the heme co-factor under conditions where folding outpaces heme delivery (Weickert et al., “Blackmore, Stabilization of Apoglobin by Low Temperature Increases Yield of Soluble Recombinant Hemoglobin in Escherichia Coli,” App. Environ. Microbiol. 63:4313-4320 (1997)). Supplementing the growth media with δ-amino levulinic acid (d-ALA), a precursor in the C5 heme biosynthesis pathway, increases levels of heme biosynthesis and thereby heme incorporation into the target protein (Kery et al., “Delta-Aminolevulinate Increases Heme Saturation and Yield of Human Cystathionine Beta-Synthase Expressed in Escherichia-Coli,” Archives Biochem. Biophys. 31:624-29 (1995); Pcsce ct al., “The 109 Residue Nerve Tissue Minihemoglobin from Cerebratulus Lacteus Highlights Striking Structural Plasticity of the Alpha-Helical Globin Fold,” Structure 10:725-735 (2002); and Summerford et al., “Bacterial Expression of Scapharca Dimeric Hemoglobin—A Simple-Model System for Investigating Protein Cooperativity,” Prot. Engineer. 8:593-599 (1995)). Increased heme biosynthesis rates through d-ALA supplementation does not achieve complete heme incorporation into all heme-binding proteins (Weickert et al., “Optimization of Heterologous Protein Production in Escherichia Coli,” Curr. Opinion In Biotechnol. 7:494-499 (1996); Shen et al., “Production of Unmodified Human Adult Hemoglobin in Escherichia-Coli,” Pro. Nat. Acad. Sci. U.S.A. 90:8108-8112 (1993); Weickert et al., “High-Fidelity Translation of Recombinant Human Hemoglobin in Escherichia Coli,” Appl. Environ. Microbiol. 64:589-1593 (1998); and Wcickert et al., “A Mutation that Improves Soluble Recombinant Hemoglobin Accumulation in Escherichia Coli in Heme Excess,” App. Environ. Microbiol. 65:640-647 (1999)).
Another technique for increasing home incorporation into recombinant proteins involves supplying the bacteria with hemin in the growth media. However, most E. coli strains do not possess an efficient heme transport system, and thus uptake of hemin relies on diffusion through the cell membrane. As a result, hemin feeding is much more effective with strains that co-express heme transport genes from other gram-negative bacteria, along with the heme-protein of interest (Graves et al., “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta—Proteins And Proteomics 1784:1471-1479 (2008); Varnado et al., “System for the Expression of Recombinant Hemoproteins in Escherichia Coli,” Prot. Exp. Pur. 35:76-83 (2004); and Varnado et al., “Expression of Recombinant Hemoproteins in E. Coli Using a Heme Protein Expression System,” Biophys. J. 384A-384A (2007)). For example, co-expression of the heme transport system from P. shigelloides, which consists of the proteins Hug A/B/C/D, TonB, and Exb B/D, while also supplementing the growth media with hemin, results in higher amounts of the target holo-protein (in this case hemoglobin) (Graves et al., “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta—Proteins And Proteomics 1784:1471-1479 (2008)). A similar method involves the co-expression of the heme receptor ChuA from E. coli. strain O157:H7 to enhance hemin (Varnado et al., “System for the Expression of Recombinant Hemoproteins in Escherichia Coli,” Prot. Exp. Pur. 35:76-83 (2004)). This latter method also shows a significant increase in the amount of heme-loaded protein generated, although in both cases, the ratio of holoprotein:apoprotein was not evaluated. Another approach utilizes the heme-permeability of E. coli strain RP523, which has the hem B, porphobilinogen synthase gene disrupted to prevent native heme synthesis. All heme and/or heme analogs are procured by the cells from the growth media and incorporation is nearly stoichiometric for proteins expressed in the cytoplasm (0.8-1.0 heme/heme analog per protein) (Woodward et al., “An Escherichia Coli Expression-Based Method for Heme Substitution,” Nat. Methods 4:43-45 (2007)).
Full incorporation of heme in recombinant proteins is also important for commercial applications. For example, the feasibility of employing recombinant human hemoglobin as an oxygen delivery pharmaceutical is limited by the yield of holoprotein that can be made in E. coli (Graves et al. “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta—Proteins And Proteomics 1784:1471-1479 (2008)). Some of the methods discussed above, while effective, require co-expression of several heme transport proteins, which could limit yields, and/or require addition of the heme cofactor.
The present invention is directed to overcoming these and other deficiencies in the art.